In view of the rapid decline in the effectiveness of antibiotics due to the emergence of resistance, there is a need for a constant supply of new compounds for effective treatment of infections. The development of antimicrobial resistance is mainly attributed to the overuse of antibiotics interfering with essential metabolic processes in bacteria. Moreover, bacteria living in biofilms, sessile cell communities embedded in a matrix of extracellular polymeric substances showing reduced metabolic activity and growth rate, exhibit up to 1000 fold higher resistance against antibiotics than free-living bacteria. Thus, novel therapeutic approaches that aim at reducing bacterial pathogenicity by interfering with bacterial virulence and biofilm formation instead of their metabolic activity are considered as highly favourable and are urgently needed.
The opportunistic pathogen Pseudomonas aeruginosa causes severe and fatal infections including such of the urinary tract, of the gastrointestinal tract, of chronic and burnt wounds, of the eyes, of the ears, and of the lungs. Its pathogenicity is strongly related to the expression of virulence factors causing progressive tissue damage and biofilm formation hindering a successful drug therapy. The regulation of pathogenicity is based on a cell-density dependent intercellular communication system known as quorum sensing (QS).
P. aeruginosa uses as signal molecules N-acyl-L-homoserine lactones (AHLs) for the las and rhl QS systems and 2-alkyl-4-(1H)-quinolones (AQs) for the pqs QS system. The latter is restricted to Pseudomonas and Burkholderia species allowing for selective therapy with pqs QS inhibitors. While Pseudomonas and Burkholderia both produce 2-heptyl-4-hydroxyquinoline (HHQ), Pseudomonas uniquely uses the Pseudomonas quinolone signal (PQS) as signal molecule. PQS and its biosynthetic precursor HHQ serve as the natural ligands and agonists of the key DNA-binding receptor PqsR. This transcriptional regulator fine-tunes a large set of genes, notably such involved in the biosynthesis of HHQ and in the production of virulence factors such as pyocyanin and lectins. Regarding biofilms, the production of extracellular DNA (eDNA) and lectins, both main biofilm matrix components, is controlled by the pqs QS system. A pqsR mutant of P. aeruginosa is pqs QS-deficient, does not produce any pyocyanin or lectin A, shows reduced eDNA production, and displays reduced pathogenicity in mice.
To date, a number of compounds have been discovered that target QS in Pseudomonas aeruginosa. The majority of these compounds has been reported to interfere with the AHL-based QS systems in Pseudomonas either via direct interaction with the receptors LasR [1, 2] or RhlR [3], at the posttranscriptional level [4-6], or at superior regulatory systems [7]. However, except an extract from Allium sativum (garlic), that exhibited no significant improvement of lung function in a clinical trial, these QS inhibitors have been only used in preclinical studies. Whereas AHL-mediated QS is widespread among Gram-negative bacteria, interference with pqs QS allows for selective therapy avoiding adverse effects on beneficial bacterial consortia present in the host. A few pqs QS inhibitors have been described acting as blockers of the signal molecule biosynthesis [8-16] or as antagonists of the receptor PqsR [17-21] A QS inhibitor based on anthranilate structure, methyl anthranilate, was shown to inhibit PQS formation and the production of the virulence factor elastase at millimolar concentrations [12]. QS inhibitors targeting the enzyme PqsA were able to reduce the production of signal molecules HHQ and PQS (IC50: ˜100 μM for 6FABA) [14, 16, 22] and enhanced the survival rate of Pseudomonas-infected mice in a thermal injury mice model [14]. However, high concentrations were necessary to obtain an in cellulo or in vivo effect. Inhibitors of the enzyme PqsD were able to reduce the biovolume of a P. aeruginosa biofilm [15], however, did not exhibit any anti-virulence properties (no effect on virulence factor pyocyanin, no effect on the survival of Pseudomonas-infected Galleria mellonella larvae; unpublished data). Zender [18] and Klein [17] reported PqsR antagonists affecting the production of virulence factor pyocyanin, however with moderate potency (IC50 values in the double-digit micromolar range). Furthermore, these compounds did not inhibit biofilm formation (unpublished data). Quinazoline-based PqsR antagonists developed in the group of Paul Williams [21] were reported to exhibit anti-virulence activity. However, the most promising compound was only moderately active in reducing pyocyanin production (IC50˜50 μM in a less PQS- and pyocyanin-producing P. aeruginosa strain). A reduction in biomass of a P. aeruginosa biofilm by this compound was observed, however at an unknown concentration. The most potent antagonist of PqsR to date—2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide—was developed in the group of Anke Steinbach and Rolf W. Hartmann [20]. 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide is highly affine to PqsR (IC50=35 nM in E. coli reporter gene assay, IC50=400 nM in P. aeruginosa reporter gene assay), strongly reduces signal molecule production (HHQ production by 54% and PQS by 37% at 15 μM), and shows excellent anti-virulence potency in cellulo (inhibition of virulence factor pyocyanin production: IC50=2 μM) and in in vivo animal infection models (strong protection of Caenorhabditis elegans nematodes and full protection of Galleria mellonella larvae from lethal P. aeruginosa infection at a concentration of 22 nM corresponding to 7.3 ng g−1 body weight). Most notably, the compound exhibited no sign of toxicity in the two animal models. However, this compound has the disadvantage of not being able to inhibit biofilm formation.
Therefore, the problem underlying the present invention is to provide novel anti-pathogenic compounds exhibiting both anti-virulence and anti-biofilm activity, especially compounds having PqsR modulating, e.g. antagonistic activity to thereby permit an effective treatment of bacterial infections.